LOCUS       sequence-482466-        4201 bp    DNA     circular SYN 08-AUG-2025
DEFINITION  Middle entry vector for mini-Golden subcloning platform; mCherry; 
            NTR-v2.
ACCESSION   .
VERSION     .
KEYWORDS    .
SOURCE      synthetic DNA construct
  ORGANISM  synthetic DNA construct
REFERENCE   1  (bases 1 to 4201)
  TITLE     Mini-Golden Subcloning Platform
REFERENCE   2  (bases 1 to 4201)
  AUTHORS   .
  TITLE     Direct Submission
  JOURNAL   Exported Aug 8, 2025 from SnapGene Server 8.0.1
            https://www.snapgene.com
COMMENT     SGRef: number: 1; type: "Journal Article"
COMMENT     Sequence Label: T2A-mCherry-P2A-NTRv2-pA-ORF-3
FEATURES             Location/Qualifiers
     source          1..4201
                     /mol_type="other DNA"
                     /organism="synthetic DNA construct"
     primer_bind     1..17
                     /label=M13 fwd
                     /note="common sequencing primer, one of multiple similar 
                     variants"
     CDS             46..99
                     /codon_start=1
                     /product="2A peptide from Thosea asigna virus capsid 
                     protein"
                     /label=T2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="EGRGSLLTCGDVEENPGP"
     CDS             100..804
                     /codon_start=1
                     /product="monomeric derivative of DsRed fluorescent protein
                     (Shaner et al., 2004)"
                     /label=mCherry
                     /note="mammalian codon-optimized"
                     /translation="VSKGEEDNMAIIKEFMRFKVHMEGSVNGHEFEIEGEGEGRPYEGT
                     QTAKLKVTKGGPLPFAWDILSPQFMYGSKAYVKHPADIPDYLKLSFPEGFKWERVMNFE
                     DGGVVTVTQDSSLQDGEFIYKVKLRGTNFPSDGPVMQKKTMGWEASSERMYPEDGALKG
                     EIKQRLKLKDGGHYDAEVKTTYKAKKPVQLPGAYNVNIKLDITSHNEDYTIVEQYERAE
                     GRHSTGGMDELYK"
     primer_bind     complement(242..260)
                     /label=mCherry-R
                     /note="mCherry, reverse primer"
     primer_bind     510..529
                     /label=mCherry-F
                     /note="mCherry, forward primer"
     primer_bind     704..727
                     /label=DsRed1-C
                     /note="DsRed1, forward primer"
     CDS             814..870
                     /codon_start=1
                     /product="2A peptide from porcine teschovirus-1 
                     polyprotein"
                     /label=P2A
                     /note="Eukaryotic ribosomes fail to insert a peptide bond 
                     between the Gly and Pro residues, yielding separate 
                     polypeptides."
                     /translation="ATNFSLLKQAGDVEENPGP"
     polyA_signal    1560..1694
                     /label=SV40 poly(A) signal
                     /note="SV40 polyadenylation signal"
     primer_bind     complement(1584..1603)
                     /label=EBV-rev
                     /note="SV40 polyA terminator, reverse primer"
     primer_bind     1638..1657
                     /label=SV40pA-R
                     /note="SV40 polyA, reverse primer"
     CDS             2131..2922
                     /codon_start=1
                     /gene="aadA"
                     /product="aminoglycoside adenylyltransferase (Murphy, 
                     1985)"
                     /label=SmR
                     /note="confers resistance to spectinomycin and 
                     streptomycin"
                     /translation="MREAVIAEVSTQLSEVVGVIERHLEPTLLAVHLYGSAVDGGLKPH
                     SDIDLLVTVTVRLDETTRRALINDLLETSASPGESEILRAVEVTIVVHDDIIPWRYPAK
                     RELQFGEWQRNDILAGIFEPATIDIDLAILLTKAREHSVALVGPAAEELFDPVPEQDLF
                     EALNETLTLWNSPPDWAGDERNVVLTLSRIWYSAVTGKIAPKDVAADWAMERLPAQYQP
                     VILEARQAYLGQEEDRLASRADQLEEFVHYVKGEITKVVGK"
     rep_origin      3015..3603
                     /direction=RIGHT
                     /label=ori
                     /note="high-copy-number ColE1/pMB1/pBR322/pUC origin of 
                     replication"
     primer_bind     3504..3523
                     /label=pBR322ori-F
                     /note="pBR322 origin, forward primer"
     primer_bind     3757..3774
                     /label=L4440
                     /note="L4440 vector, forward primer"
     terminator      complement(3933..3960)
                     /label=rrnB T2 terminator
                     /note="transcription terminator T2 from the E. coli rrnB 
                     gene"
     terminator      complement(4052..4138)
                     /gene="Escherichia coli rrnB"
                     /label=rrnB T1 terminator
                     /note="transcription terminator T1 from the E. coli rrnB 
                     gene"
     primer_bind     join(4187..4201,1..8)
                     /label=M13/pUC Forward
                     /note="In lacZ gene"
     primer_bind     join(4201,1..17)
                     /label=M13 Forward
                     /note="In lacZ gene. Also called M13-F20 or M13 (-21) 
                     Forward"
ORIGIN
        1 gtaaaacgac ggccagtctt aagctcgggc ccggtctctt cgcccgaggg cagaggcagt
       61 ctgctgacat gcggtgacgt ggaagagaat cccggccctg tgagcaaggg cgaggaggac
      121 aacatggcca tcatcaagga gttcatgcgc ttcaaggtgc acatggaggg ctccgtgaac
      181 ggccacgagt tcgagatcga gggcgagggc gagggccgcc cctacgaggg cacccagacc
      241 gccaagctga aggtgaccaa gggcggcccc ctgcccttcg cctgggacat cctgtcccct
      301 cagttcatgt acggctccaa ggcctacgtg aagcaccccg ccgacatccc cgactacttg
      361 aagctgtcct tccccgaggg cttcaagtgg gagcgcgtga tgaacttcga ggacggcggc
      421 gtggtgaccg tgacccagga ctcctccctg caggacggcg agttcatcta caaggtgaag
      481 ctgcgcggca ccaacttccc ctccgacggc cccgtaatgc agaagaagac catgggctgg
      541 gaggcctcct ccgagcggat gtaccccgag gacggcgccc tgaagggcga gatcaagcag
      601 aggctgaagc tgaaggacgg cggccactac gacgccgagg tcaagaccac ctacaaggcc
      661 aagaagcccg tgcagctgcc cggcgcctac aacgtcaaca tcaagctgga catcacctcc
      721 cacaacgagg actacaccat cgtggaacag tacgagcgcg ccgagggccg ccactccacc
      781 ggcggcatgg acgagctgta caagggaagc ggagctacta acttcagcct gctgaagcag
      841 gctggagacg tggaggagaa ccccgggcct atgactattg ttcaagctgc ccaatcccgc
      901 tactccacca aagcctttga tgcttcgcgc aaattgcctg aagagaaagt cgcggcagtg
      961 aaagagttaa tccgcatgag tgcgtccagt gtcaactcgc aaccttggca ttttattgtc
     1021 gcgagcagtg aagagggaaa agcgcgcatc gccaaagcaa cacaaggtgg ttttgctgct
     1081 aatgagcgca agattttgga tgcttctcac gttgtggtgt tctgcgccaa aacggcgatt
     1141 gatgaagcgt acctactcga ccttttggaa agcgaagaca aagatggccg ctacgccgat
     1201 gtcgaagcaa aaaatggcat gcacgctggt cgttcatttt tcgtcaacat gcaccgcttt
     1261 gacttgaaag acgcgcatca ctggatggaa aagcaagttt acctcaatgt ggggacgctg
     1321 ctattgggtg cttctgcgat ggagatcgac gcggtgccaa ttgaaggctt cgatgccaag
     1381 gtgcttgatg aggagtttgg tctgcgtgag aagggcttta ccagcgtggt gattgtgccg
     1441 ctgggttacc atagcgaaga cgattttaat gctaagctgc caaaatcacg ttggtcagca
     1501 gagactgttt ttaccgaaat ctaactcgag tctagaacta tagtgagtcg tattacgtag
     1561 atccagacat gataagatac attgatgagt ttggacaaac cacaactaga atgcagtgaa
     1621 aaaaatgctt tatttgtgaa atttgtgatg ctattgcttt atttgtaacc attataagct
     1681 gcaataaaca agttaacaac aacaattgca ttcattttat gtttcaggtt cagggggagg
     1741 tgtgggaggt tttttaattc ctcaagagac ctctagacca gccaggacag aaatgcctcg
     1801 acttcgctgc tacccaaggt tgccgggtga cgcacaccgt ggaaacggat gaaggcacga
     1861 acccagtgga cataagcctg ttcggttcgt aagctgtaat gcaagtagcg tatgcgctca
     1921 cgcaactggt ccagaacctt gaccgaacgc agcggtggta acggcgcagt ggcggttttc
     1981 atggcttgtt atgactgttt ttttggggta cagtctatgc ctcgggcatc caagcagcaa
     2041 gcgcgttacg ccgtgggtcg atgtttgatg ttatggagca gcaacgatgt tacgcagcag
     2101 ggcagtcgcc ctaaaacaaa gttaaacatt atgagggaag cggtgatcgc cgaagtatcg
     2161 actcaactat cagaggtagt tggcgtcatc gagcgccatc tcgaaccgac gttgctggcc
     2221 gtacatttgt acggctccgc agtggatggc ggcctgaagc cacacagtga tattgatttg
     2281 ctggttacgg tgaccgtaag gcttgatgaa acaacgcggc gagctttgat caacgacctt
     2341 ttggaaactt cggcttcccc tggagagagc gagattctcc gcgctgtaga agtcaccatt
     2401 gttgtgcacg acgacatcat tccgtggcgt tatccagcta agcgcgaact gcaatttgga
     2461 gaatggcagc gcaatgacat tcttgcaggt atcttcgagc cagccacgat cgacattgat
     2521 ctggctatct tgctgacaaa agcaagagaa catagcgttg ccttggtagg tccagcggcg
     2581 gaggaactct ttgatccggt tcctgaacag gatctatttg aggcgctaaa tgaaacctta
     2641 acgctatgga actcgccgcc cgactgggct ggcgatgagc gaaatgtagt gcttacgttg
     2701 tcccgcattt ggtacagcgc agtaaccggc aaaatcgcgc cgaaggatgt cgctgccgac
     2761 tgggcaatgg agcgcctgcc ggcccagtat cagcccgtca tacttgaagc tagacaggct
     2821 tatcttggac aagaagaaga tcgcttggcc tcgcgcgcag atcagttgga agaatttgtc
     2881 cactacgtga aaggcgagat caccaaggta gtcggcaaat aaccctcgag ccacccatga
     2941 ccaaaatccc ttaacgtgag ttacgcgtcg ttccactgag cgtcagaccc cgtagaaaag
     3001 atcaaaggat cttcttgaga tccttttttt ctgcgcgtaa tctgctgctt gcaaacaaaa
     3061 aaaccaccgc taccagcggt ggtttgtttg ccggatcaag agctaccaac tctttttccg
     3121 aaggtaactg gcttcagcag agcgcagata ccaaatactg ttcttctagt gtagccgtag
     3181 ttaggccacc acttcaagaa ctctgtagca ccgcctacat acctcgctct gctaatcctg
     3241 ttaccagtgg ctgctgccag tggcgataag tcgtgtctta ccgggttgga ctcaagacga
     3301 tagttaccgg ataaggcgca gcggtcgggc tgaacggggg gttcgtgcac acagcccagc
     3361 ttggagcgaa cgacctacac cgaactgaga tacctacagc gtgagctatg agaaagcgcc
     3421 acgcttcccg aagggagaaa ggcggacagg tatccggtaa gcggcagggt cggaacagga
     3481 gagcgcacga gggagcttcc agggggaaac gcctggtatc tttatagtcc tgtcgggttt
     3541 cgccacctct gacttgagcg tcgatttttg tgatgctcgt caggggggcg gagcctatgg
     3601 aaaaacgcca gcaacgcggc ctttttacgg ttcctggcct tttgctggcc ttttgctcac
     3661 atgttctttc ctgcgttatc ccctgattct gtggataacc gtattaccgc ctttgagtga
     3721 gctgataccg ctcgccgcag ccgaacgacc gagcgcagcg agtcagtgag cgaggaagcg
     3781 gaagagcgcc caatacgcaa accgcctctc cccgcgcgtt ggccgattca ttaatgcagc
     3841 tggcacgaca ggtttcccga ctggaaagcg ggcagtgagc gcaacgcaat taatacgcgt
     3901 accgctagcc aggaagagtt tgtagaaacg caaaaaggcc atccgtcagg atggccttct
     3961 gcttagtttg atgcctggca gtttatggcg ggcgtcctgc ccgccaccct ccgggccgtt
     4021 gcttcacaac gttcaaatcc gctcccggcg gatttgtcct actcaggaga gcgttcaccg
     4081 acaaacaaca gataaaacga aaggcccagt cttccgactg agcctttcgt tttatttgat
     4141 gcctggcagt tccctactct cgcgttaacg ctagcatgga tgttttccca gtcacgacgt
     4201 t
//
